Polymer surfactants for gene therapy applications

ABSTRACT

A composition and method capable of delivering pharmaceutical or biomedical materials includes a tri-block surfactant having a hydrophilic block, a charged water-soluble block and a hydrophobic block.

This invention was supported by Grant No. DMR 9984102 from the NationalScience Foundation and Grant No. 1HG01386-07 from the National HumanGenome Research Institute, both of which may have rights in theinvention.

BACKGROUND OF THE INVENTION

This invention relates to a composition and method capable of deliveringpharmaceutical or biomedical materials including a tri-block surfactanthaving a hydrophilic block, a charged water-soluble block andhydrophobic block. The tri-block surfactant provides a biodegradablecarrier for pharmaceutical or biomedical materials.

Viral vectors have encountered serious safety concerns, such asinflammatory reactions and virus replication. In contrast, non-viralvectors, although markedly lower in inefficiency at the present time,offer flexibility in design and great potential in meeting requirementssuch as receptor recognition, improved safety profile, protection of DNAfrom degradation by nucleases and improved immunogenicity for genetherapy applications. While many attempts have been made to developnon-viral therapy gene delivery systems by using cationic liposomes orpolymers and combinations thereof, the results of such efforts have notbeen very successful.

Cationic polymers, such as poly-L-lysine (PLL), poly (ethylenimine)(PEI), poly(dimethylaminoethylmethacrylate) (PDMAEMA),diethylaminoethyl-dextran and chitosan, have been used in complexationstudies, with the positively charged PEI considered to be the mostpromising delivery candidate. Linear PEI is marketed commercially as atransfection agent under the trademark ExGen 500. Unfortunately, theDNA-PEI complex is only partly soluble in an aqueous environment. Moreimportantly, PEI is quite toxic.

In the search for effective drug delivery and DNA delivery carriers,synthetic polymers and hybrids with natural polymers have been studied,including biodegradable polymers and their copolymers as well as the useof core-shell nanoparticles.

Drug and DNA delivery and release behavior is governed by the ability ofthe carrier to pass through lipophilic cell membranes (transfection).Bioactive agents such as proteins and DNA may lose their bioactivitieswhen exposed to hydrophobic surfaces and to degradation products ofpolymers.

SUMMARY OF THE INVENTION

It is an object of the invention to provide a non-viral carrier forpharmaceutical and biomedical materials.

Another object of the invention is to provide a nonviral carrier whichcan transport intact pharmaceutical and biomedical materials across cellmembranes.

These and other objects of the invention are achieved by providing asurfactant comprising a triblock copolymer including a hydrophilicblock, a charged water-soluble block and a hydrophobic block. Thesurfactant can form complexes with pharmaceutical or biomedicalmaterials and transport these materials across cell membranes.

If the pharmaceutical or biomedical material is positively charged, thecharged water-soluble block is negatively charged, while a positivelycharged water-soluble block will be used if the biomedical orpharmaceutical material is negatively charged.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of a synthesis of a linear polyethyleneglycol-polylactide-polylysine tri-block copolymer surfactant inaccordance with the invention;

FIG. 2 is an illustration of a synthesis of a linear polyethyleneglycol-polylysine-polylactide tri-block polymer surfactant in accordancewith the invention;

FIG. 3 is an illustration of a synthesis of a tri-arm block polymersurfactant in accordance with the invention;

FIG. 4(a), (b), (c) are schematic illustrations of (a) micelle formationof a tri-block polymer surfactant in a hydrophilic environment, (b) atri-arm tri-block copolymer (c) micelle formation of a tri-blockcopolymer in a hydrophobic environment;

FIG. 5(a) is a schematic illustration of a complex with DNA, 5(b) is aschematic illustration of condensed DNA encapsulated by the tri-blocksurfactant where the condensed DNA forms a supramolecular complex withoppositely charged blocks. The surface of the complex encapsulates theDNA and the other two blocks, one hydrophilic and one hydrophobic, makethe supramolecular assembly soluble in either hydrophilic or hydrophobicenvironment;

FIG. 6 is a schematic illustration of a transport of a DNA surfactantcomplex across a cell membrane; and

FIG. 7 is a schematic illustration of disassembly of the DNA-surfactantcomplex inside the cell.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Polyelectrolyte-surfactant complexes (PSC's) are unique materials withthe ability to spontaneously self-assemble into highly orderednanostructures. PSC's are formed by the complex formation ofpolyelectrolytes and oppositely charged ionic surfactants, usually inaqueous solution. Many biologically active materials such as DNA,polypeptides, polysaccharides, etc. are natural polyelectrolytes.

PSCs can be considered as microphase-separated materials, showingsegregation into polar and hydrophobic domains on a nanometer lengthscale. The microphase separation can and often does lead to long-rangeorder, so that these PSCs form ordered crystal-like structures,macro-lattices, with constants of typically a few nanometers.

In other, less-ordered PSCs, discrete microphase-separated complexes areformed, usually but not always spherical in shape. The less ordered PSCsare not arranged on a three dimensional lattice, but only show aliquid-like short range order, or when sufficiently diluted, no orderedarrangement at all. This type of PSC is referred as colloidalpolyelectrolyte surfactant complexes (CPSCs).

Parameters influencing the structure and morphologies of PSCs (andCPSCs) include the chain length, charge density, crosslinker density,backbone hydrophobicity and persistence length of the polyelectrolyte;the nature of the solvent, the chemical composition of the solvent,whether the solvent is a pure fluid or mixture, the physical propertiesof the solvent including dielectric constant and viscosity; with respectto the surfactant, the number and type of head charges, geometry ofhydrophobic tail(s), chain length, geometry of hydrophilic part,topology, i.e. single or double tail; and external parameters such aspH, ionic strength, PSC concentration, temperature, pressure, nature ofcounterion and stoichiometry.

In the present invention a polyelectrolyte and tri-block surfactant forma polyelectrolyte surfactant complex which can cross a cell membrane anddeliver the essentially encapsulated polyelectrolyte to the cell withoutsignificant loss of the polyelectrolyte's bioactivity. Preferably thePSC is a CPSC.

The tri-block copolymer includes hydrophilic, charged and hydrophobicblocks. Preferably the blocks are FDA approved and the hydrophobic blockis biodegradable. The presence of a hydrophilic block and a hydrophobicblock ensures solubility of the tri-block copolymer in both the aqueousenvironment and hydrophobic environment. The presence of the hydrophobicblock can also be used to modify the surface properties of the PSC andthereby further protect the PSC components in the supramolecularstructure. The biodegradable nature of the hydrophobic block can beselected to permit degradation after the soluble supramolecular complexcrosses the cell membrane.

Examples of suitable hydrophilic block components include but are notlimited to polyethylene oxide, polyethylene glycol (PEG) or otherwater-soluble neutral polymers, such as polypropylene oxide at lowtemperatures.

Examples of suitable charged block components include but are notlimited to poly(aminoacids), polyacrylic acid, polyethylenemine andpoly(dimethylaminoethyl-methacrylate), as well as polysaccharides,including but are not limited to chitin/chitosan and hyaluronic acid.Examples of poly(aminoacids) include but are not limited topoly(L-lysine), poly(diethylamino-L-glutamine), polyarginine andpolyornithine.

Examples of suitable hydrophobic components include but are limited topoly(glycolide), poly(lactide), poly(lactide-co-glycolide),polyanhydride, poly(dioxanone), sebacic acid, poly(ε-caprolactone), andpolyhydroxybutyrate as well as polyalkylene oxide, e.g., polypropyleneoxide at high temperatures. Polypropylene oxide is hydrophilic at lowtemperatures and hydrophobic at high temperatures.

Any pharmaceutical or biomedical agent which is charged can be includedin the PSC. By “pharmaceutical or biomedical agent” is meant abiologically active molecule that can be used in the treatment, cure,prevention or diagnosis of disease or is otherwise used to enhancephysical or mental well being in humans or other animals. Thebiologically active molecules include but are not limited to proteins,peptides, oligonucleotides, DNA, RNA and polysaccharides and of courseany other molecules having such activity.

Proteins and peptides which may be used in accordance with the presentinvention include enzymes such as proteases (e.g. bromelain, papain,collagenase, elastase), lipases (e.g. phospholipase C), esterases,glucosidases, hyaluronidase, exfoliating enzymes; antibodies andantibody derived actives, such as monoclonal antibodies, polyclonalantibodies, single chain antibodies and the like; reductases; oxidases;peptide hormones; natural structural skin proteins, such as elastin,collagen, reticulin and the like; anti-oxidants such as superoxidedismutase, catalase and glutathione; free-radical scavenging proteins;DNA-repair enzymes, for example T4 endonuclease 5 and P53; antimicrobialpeptides, such as magainin and cecropin; a milk protein; a silk proteinor peptide; and any active fragments, derivatives of these proteins andpeptides; and mixtures thereof an anti-viral agent (such as acyclovir);an anti-hemorrhoid compound, an anti-wart agent (such aspodophyllotoxin) and a plant extract and mixtures thereof.

Cytokines can also be incorporated into the delivery system. Thecytokines include vascular endothelial growth factor (VEGF), endothelialcell growth factor (ECGF), fibroblast growth factor (FGF), insulin-likegrowth factor (IGF), bone morphogenic growth factor (BMP),platelet-derived growth factor (PDGF), epidermal growth factor (EGF),thrombopoietin (TPO), interleukins (IL1-IL15), interferons (IFN),erythropoietin (EPO), ciliary neurotrophic factor (CNTF), colonystimulating factors (G-CSF, M-CSF, GM-CSF), glial cell-derivedneurotrophic factor (GDNF), leukemia inhibitory factor (LIF), andmacrophage inflammatory proteins (MIP-1a,-1b,-2).

Genetic material can also be incorporated in the delivery system. Genetherapy can be used to introduce an exogenous gene in an animal tosupplement or replace a defective or missing gene. For example, genesincluding but not limited to, genes encoding for HLA-B, insulin,adenosine deaminase, cytokines and coagulant factor VIII can beincorporated into the matrix and released over a fixed time period. Thedesired material can be operably linked to a variety of promoters wellknown in the art. Examples of promoters include, but are not limited to,an endogenous adenovirus promoter, such as the E1 a promoter or the Ad2major late promoter (MLP) or a heterologous eucaryotic promoter, forexample a phosphoglycerate kinase (PGK) promoter or a cytomegalovirus(CMV) promoter. Similarly, those of ordinary skill in the art canconstruct adenoviral vectors using endogenous or heterologous poly Aaddition signals.

In an alternate embodiment the tri-block surfactant encapsulates thedesired gene when it is in a condensed state by using kinetic processingunder non-equilibrium conditions. The resultant supramolecular complexwith a condensed DNA core has a duel amphiphilic property and istargeted specifically to overcome challenges in the solubility of thosecomplexes under a variety of aqueous and hydrophobic solventenvironments, the enhancement of gene transfection and gene protection,as well as the final gene delivery to the nucleus in the cell.

The DNA can first be collapsed into a condensed state by dissolving orsuspending DNA chains in a poor or non-solvent. For example, the DNA canbe condensed in a solvent mixture of 94% w/w % N,N-dimethyl formamideand 6% w/w % water. In coil-to-globule transition phenomena volumechanges of the order of thousands can be observed. There will be acompetition between the formation of globules or other condensed statesand the aggregation of DNA molecules. Thus, kinetic processing undernon-equilibrium conditions will be considered.

The condensed DNA molecule(s) can be coated with apolyelectrolyte-surfactant complex shell using the strong electrostaticinteractions between the DNA and the tri-functional surfactant thatcontains an oppositely charged block. This complex formation involvesanother volume contraction that could reduce the supramolecular complexto even smaller sizes. It is noted that DNA-surfactant complexes aregenerally insoluble. However, each surfactant molecule is covalentlyattached to both a hydrophilic block and a hydrophobic block. Thus, thesupramolecular complex can be designed to be soluble in both thehydrophobic environment needed for the DNA condensation, encapsulation,and cell membrane penetration and the aqueous environment needed forgene delivery and movements inside the cell.

The following examples illustrate preparation of a tri-block surfactantin accordance with the invention, the formation of complexes with DNA,transfection of cells and disassembly of the complex.

The tri-block surfactant can be linear ELB or EBL where E=hydrophilicblock, L=positively or negatively charged block and B=hydrophobic block.In addition, the notation EBL implies that after biodegradation, E and Lare no longer covalently linked together, while ELB implies that E and Lremain covalently bonded after biodegradation. The morphology of thetri-arm star ELB indicates that after biodegradation, E and L willremain covalently linked. Preferably, the PEG used in the followingexamples has a molar mass of less than 20 k.

EXAMPLE 1 Synthesis of Biodegradable Tri-Block Copolymer PEG-b-PLA-PLL

PLA-b-PEG could be synthesized by polymerization of lactide in thepresence of PEG as described in Zhu et al. J. Appl. Polym. Sci., 39, 1-9(1990). The hydroxyl end group of the diblock polymers can then beconverted to an amino group by reaction with N-CBZ-glycine as set forthin C. Y. Won et al., J. Appl. Polym. Sci., 70, 953-963 (1998), andsubsequent deprotection. The terminal amino group initiates thepolymerization of NCA to give the tri-block copolymer in accordance withKricheldorf, “Aminoacid-N-carboxy-anhydride and related heterocycles”Spinger-Vertag, Berlin (1987) pp. 3-58. NCA is not commerciallyavailable. This synthesis is shown in FIG. 1.

EXAMPLE 2 Synthesis of Tri-Block Copolymer PEG-PLL-PLA

Amino-terminated PEG would initiate polymerization of NCA as describedby Kricheldorf, which would yield PEG-b-N-CBZ-PLL. The end-amino groupfurther initiates the polymerization of lactide resulting in ELB(“E”=hydrophilic block, “L”=charged block, and “B”=hydrophobic block).After deprotection of the N-CBZ group, the tri-block copolymerPEG-b-PLL-b-PLA could be obtained. This synthesis is shown in FIG. 2.

EXAMPLE 3 Tri-Arm Star Block Synthesis

The first step is to modify the PEG end groups with N-CBZ serine so thatPEG carries two different functional groups, one being the hydroxylgroup and the other being the protected amino group. The second step isto use the hydroxyl group of the modified PEG to initiate thepolymerization of lactide using Sn(Oct)₂ as catalyst in order to obtainthe two-arm block copolymer of PEG and PLA with the remaining protectedamino group. The third step is to deprotect the N-CBZ group of thetwo-arm block copolymer in order to activate the amino group. Finally,the protected polylysine is conjugated to the copolymer by thering-opening polymerization of NCA with the amino group as an initiator.The star three-arm block copolymer is obtained by deprotecting thependant N-CBZ group. This synthesis is shown in FIG. 3.

EXAMPLE 4 Transfection of DNA Material

A schematic diagram of a micelle of 6 tri-block polymers FIG. 4(a) inequilibrium with an tri-arm star tri-block polymer FIG. 4(b) and themicelle formed in a hydrophobic environment is shown in FIG. 4(c). Asshown in FIG. 5(a), the micelles are contacted with DNA strands in ahydrophilic environment and form a surfactant-DNA complex. Thismicelle-DNA complex in an aqueous environment is undersirable.Accordingly, preferably the DNA will first be condensed in a poorsolvent, relatively hydrophobic solvent, and then the condensed DNA willbe contacted with the surfactant, i.e. below its critical micelleconcentration, in the poor, relatively hydrophobic, environment so thata strong DNA-charged block complex shell is formed on the surface of thecondensed DNA. This supramolecular complex has both hydrophobic andhydrophilic blocks covalently bonded to the complex and is soluble ineither the hydrophobic or the hydrophilic environment. Such asupramolecular complex can then become a soluble complex in the aqueousenvironement, as shown in FIG. 5(b). In order to avoid aggregation, itis permissible to neutralize amounts of both positive charges from thesurfactant and negative charges from the polyeletrolyte in the CPSC soas to avoid further aggregation of the CPSC.

Transfection is schematically illustrated in FIG. 6. The duality insolubility of the CPSC should promote particle transmission through thecell membrane. In the hydrophobic region of the cell membrane, theB-chains should extend while the E-chains should collapse. The chainextensions and contractions will increase the transfection efficiency.

The disassembly of CPSC is shown in FIG. 7. Inside the cell, thehydrophilic E and hydrophobic B regions on the surface of the coreshould provide better protection for DNA from attack by nucleases. Withbiodegradation of the B-chains, the CPSC will destabilize. This processshould provide easier access of the DNA by the nucleus. The positivelycharged L-chains are still covalently bound to the hydrophilic E-chainsmaking these components less toxic and easier for discharge from thecell.

EXAMPLE 5 Synthesis of N2.N6-bis((phenylmethoxy)carbonyl)-L-lysine(2)

This synthesis is in accordance with Galbiati, B.; Ferrario, T.; Merli,V. WP 0110851(2001). A 250 mL three-necked flask was loadedconsecutively with water (20 mL), 1,4-dioxane (20 mL) and L-lysine (2.1g, 14.4 mmol). The mixture was stirred until complete dissolution. ThepH was adjusted to about 10.5 by addition of 30% NaOH.Benzylchloroformate (5.2 g, 30.6 mmol) was added while maintaining thepH at about 10˜11 by adding at the same time 30% NaOH. At the end of theaddition, the reaction was stirred at 20° C. for about 1 hour. The pHwas adjusted to 5 with 37% HCl. Ethyl acetate (30 mL) was added and thepH was adjusted to 1 with 37% HCl. The mixture was stirred at roomtemperature for about 30 minutes, the organic layer was separated andthe aqueous layer was extracted with ethyl acetate (20 mL). The combinedorganic layer was washed with brine (30 mL), and dried over Na₂SO₄.Then, the solvent was evaporated to yield a yellowish oil (6.0 g, 99%).The oil was pre-dried by azeotropic distillation with benzene.

EXAMPLE 6 Synthesis of N5-((phenylmethoxy)carbonyl)-L-lysine,N-carboxyanhydride (NCA)

This synthesis was performed in accordance with Cannata, V.; Merli, V.;Sagwatti, S. EP 943621 (1999). Compound 2 (6.0 g, 14 mmol) was dissolvedin dichloromethane (40 mL). To this solution, dimethyl formamide, DMF(1.5 mL) was added. The mixture was cooled to 0° C. Under stirring,thionyl chloride (2.32 g, 19 mmol) was added during 15 minutes. Thereaction mixture was kept for one hour at 0° C., and then at 10° C. fora further 2 hours. After that it was evaporated under vacuum, andfurther dried in vacuum at 40-50° C. for 12 hours. A yellow-orange oil(4.5 g, 99%) was obtained. Compound NCA was obtained in this way at apurity of 90% as deteremined by NMR spectroscopy.

The tri-functional surfactant of the invention has a hydrophilic block(E), a charged block (L) and an additional biodegradable and flexiblehydrophobic block (B). This third biodegradable hydrophobic (B) blockcan serve at least four useful functions. (1) It can modify thehydrophobic surface of the DNA-surfactant complex segments. (2) Thesupramolecular complex can be designed to be soluble not only in theaqueous environment but also in the hydrophobic environment. An increasein the compatibility with the interior of bilayer cell membranes shouldalso promote the penetration of such complexes across the cell membrane.(3) The presence of a duality of hydrophobic and hydrophilic chains onthe complex surface could increase the protection of genes in thesupramolecular core. (4) The biodegradable block can be designed todestabilize the complex for eventual release of entrapped DNA chains.

The above description is illustrative and not limiting. Furthermodifications will be apparent to one of ordinary skill in the art inlight of the disclosure and appended claims.

1. A surfactant comprising a triblock copolymer including a hydrophilicblock, a charged water-soluble block and a hydrophobic block.
 2. Asurfactant according to claim 1 wherein the charged water-soluble blockis selected from the group consisting of a polyamino acid, chitin,chitosan, and polysaccharide.
 3. A surfactant according to claim 2wherein the polyamino acid is selected from the group consisting ofpoly(lysine), poly(arginine) and poly(ornithine).
 4. A surfactantaccording to claim 1 wherein the hydrophobic block is selected from thegroup consisting of polylactide, polylactide-co-glycolide,poly(dioxanone), polyanhydride, sebacic acid, poly(ε-caprolactone),chitin, chitosan, poly(hydroxybutyrate) and poly(glycolide).
 5. Asurfactant according to claim 1 wherein the hydrophilic block isselected from the group consisting of polyethylene oxide, polyethyleneglycol and polypropylene oxide.
 6. A composition comprising a surfactantcomplex according to claim 1 and at least one selected from the groupconsisting of RNA and DNA.
 7. A composition comprising a surfactantaccording to claim 1 and a pharmaceutical.
 8. A method of delivering DNAto a tissue comprising forming a complex including a surfactantaccording to claim 1 and DNA, and contacting the tissue with thecomplex.